Many viruses evade host defense mechanisms by targeting specific host vulnerabilities, revealing critical points in host pathways regulating antiviral responses. As an example, the NS1 protein of influenza virus, a major virulence factor, inhibits host gene expression and signal transduction required to mount innate and adaptive immune responses. In infected cells, NS1 is localized in the nucleus and in the cytoplasm. Based on the knowledge of NS1 functions in the nucleus as an inhibitor of gene expression, we performed a high throughput screen of 200,000 synthetic chemical compounds. We have identified eight classes of novel compounds that significantly restored gene expression in the presence of NS1 and also significantly inhibited both influenza virus replication and viral-mediated cell death. Two of these compounds inhibit the replication of viruses other than influenza virus, indicating that they either initiate a host antiviral response or inactivate a host factor required for virus replication. We will combine chemical biology, cell biology, and virology to investigate the mechanisms by which these compounds inhibit virus replication with the twin goals of identifying novel therapeutic targets and also improving the characteristic of the compounds as potential leads to novel molecular therapy. We will pursue the following aims: Aim 1. To investigate structure-activity relationship (SAR) of antiviral compounds. The UTSW Synthetic Chemistry Laboratory will produce targeted libraries (~15-25 analogs) based upon the best of our compounds, which are prioritized according to their potency to inhibit virus replication and their lack of cytotoxicity. We will conduct quantitative structure-activity analysis to establish a pharmacophore model of each compound, to provide an understanding of key elements affecting potency. Aim 2. To address issues of ADME of antiviral compounds. We will test our best compound series for, solubility, cellular absorption, metabolism and cellular toxicity. This information will be important to study these inhibitors in mice infected with influenza virus as proposed in Aim 3. We will make affinity probes from compounds that are active in the nanomolar range for a biochemical approach to target identification. Aim 3. To determine the activity of NS1 inhibitors on Antiviral Responses in vivo. We will use mice models of influenza virus infection for preclinical tests of compound efficacy in preventing infection. The mouse model will be used to measure the effects of compounds on virus replication in lungs and on the severity of disease. Interferon response will also be evaluated. In addition, mice will be infected with VSV, vaccina virus, and influenza A and B viruses to investigate compound specificity. In sum, these studies will likely reveal novel leads for antiviral therapies as well as provide information on novel mechanisms of viral-host interactions and pathways.